Human infections by the intracellular bacterial pathogens Chlamydia trachomatis and C pneumoniae present an enormous health care problem. Infections by these pathogens have been associated with engendering and/or exacerbating several chronic diseases, and some of these Chlamydial infections have proved to be refractory to antibiotic therapy. The lack of therapeutic efficacy results from the attenuated metabolic rate of infecting chlamydiae under some circumstances, in combination with the modest intracellular concentrations achievable by normal delivery of such drugs to the inclusions within which chlamydiae reside in the host cell cytoplasm. The major therapeutic goal of the studies outlined here is to develop a means by which antibiotics or other therapeutic agents can be delivered in a targeted manner to the intracellular Chlamydial inclusion at effective concentrations, without toxicity to the host cell or infected tissue. Our initial studies indicate that Chlamydial infection elicits increased expression of host cell folic acid receptors (FAR), and that folic acid-conjugated nanoparticles may provide a novel and highly effective means of intracellular delivery of therapeutic agents to Chlamydia- infected cells. The working hypothesis underlying the studies proposed is that host cells infected with either C trachomatis or C pneumoniae can be cleared of actively- or persistently-infecting organisms via nanoparticle-mediated targeted delivery of effective concentrations of antibiotics known to work against active Chlamydial infections. We further hypothesize that nanoparticle-facilitated delivery will require reduced amounts of therapeutic materials, which could engender significant health care cost reductions. To pursue that end, three Specific Aims will be addressed. In Aim One, we will define the time-course of expression of folate receptors following C trachomatis infection of a relevant panel of human host cells in culture and in a mouse model of C trachomatis infection, and define the localization of folic acid receptors between the host cell and cytoplasmic inclusion membranes. In Aim Two, we will determine at what time point post-in vitro infection delivery of those nanoparticles is most effective, and whether delivery of antibiotics to infected cells by nanoparticles is effective in clearing C trachomatis infection. In addition, using fluorescence microscopy, we will study the intracellular trafficking of nanoparticles in infected and uninfected cells to understand the mechanism of nanoparticle targeting to inclusions. In Aim Three, using in vivo imaging, molecular genetics, and quantitative analytical methods, we determine if FAR targeted delivery of nanoparticle antibiotics to Chlamydia-infected mice clears synovial infection in vivo. If successful, results of this research will suggest a novel nanotechnology-based therapeutic regimen for effective treatment of an important health care problem. PUBLIC HEALTH RELEVANCE: Chlamydiae are associated with several chronic diseases, including inflammatory arthritis, tubal occlusion leading to ectopic pregnancy, and cervical cancer (C trachomatis); C pneumoniae has been compellingly associated with atherosclerosis, inflammatory arthritis, and temporal arteritis, among several others. The proposed research is expected to positively affect human health, because, it is expected to lead to the development of an effective approach for the treatment of persistent Chlamydial infections.